Multi-drug Resistant Tuberculosis (MDR-TB) is an emerging infection of Mycobacterium tuberculosis resistant to two or more drugs which is difficult to treat, and in some cases untreatable. New vaccines are urgently needed. This Project will generate a new series of live attenuated M. tuberculosis strains that have been rendered avirulent and more immunogenic through a series of targeted mutations. The starting strain contains the original attenuating mutation found in BCG, the only existing TB vaccine. To make the strain safer, a deletion of the genes required for the synthesis of the vitamin pantothenate is added. This vaccine is not only safer than BCG, but protects both immunocompetent mice and CD4-deficient mice, a model for HIV infection, against aerosolized M. tuberculosis challenge. To make the strain more immunogenic, we are incorporating a number of mutations that when deleted alter the interaction with the host immune response and are postulated to enhance immunogenicity. One class has been shown to alter the mycolic acids so that these mycolic acids do not induce typical granuloma formation and induction of TNF-alpha. The second class mediates enhanced apoptosis, which should enhance the elicitation of MHC Class I antigen presentation. The third class of mutation fails to induce IL-12, an important cytokine required to induce an optimal effective immune response. Preclinical studies using the mouse model of tuberculosis, in collaboration with the other three projects, will be carried out to assess safety and immunogenicity, and to determine the immunologic correlates of protection against infection and disease.